Most mature blood cells are short lived and must be replaced continuously throughout adult life. Lifelong production of mature blood cells depends on the activity of a small pool of multipotential hematopoietic stem cells (HSC) located mainly in the bone marrow. These stem cells are capable of self-renewal and the generation of progenitor cells committed irreversibly to one of three main progenitor cell types: erythroid, myeloid and lymphoid. Erythroid progenitor cells give rise to erythrocytes. The myeloid lineage produces neutrophils and monocytes as well as platelets, mast cells, eosinophils and basophils. The lymphoid lineage gives rise to B and T lymphocytes and NK cells.
The ability of stem cells to undergo substantial self-renewal as well as the ability to proliferate and differentiate into all of the hematopoietic lineages makes stem cells the target of choice for a number of gene therapy applications. Successful gene transfer into stem cells should provide long-term repopulation of an individual with the modified cells and their progeny, which will express the desired gene product. There are many diseases that affect hematopoietic cells for which gene therapy and/or bone marrow transplantation could be useful to alleviate or cure the disease. Such diseases include severe combined immunodeficiency (SCID), chronic myelogenous leukemia (CML), .beta.-thalassemia, sickle cell anemia and the like. Since blood cells have a finite life cycle, gene transfer into more mature hematopoietic cells, such as T cells, at best, provides only transient therapeutic benefit. For example, a SCID patient was treated by introducing a normal ADA gene into her lymphocytes ex vivo and reinjecting the transduced lymphocytes back into the patient (Biotechnology News (1993) 13:14). For effective therapy, ADA-carrying lymphocytes had to be reinjected into the patient every six months. Introducing the ADA gene into HSCs could obviate repeated treatments since the ADA-carrying stem cells could repopulate the bone marrow and completely cure the disease. Thus, gene therapy efforts are focused on hematopoietic stem cells because the transduction and transplantation of these cells would provide a means of ensuring a continuous supply of genetically modified hematopoietic cells during the lifetime of the patient. Hematopoietic stem cells are also ultimately responsible for restoring blood cell numbers if the hematopoietic system is depleted in some way.
DNA and retroviruses as well as other types of nucleic acid delivery vehicles, have been used to transfer genes in gene therapy. The use of retroviral vectors to mediate gene therapy is preferred over other vectors for nucleic acid transduction because of their high transduction efficiency. Other advantages include the reduced possibility of gene rearrangement and the single or low copy number transfer of the gene of interest. However, transducing HSCs presents a challenge because the stem cells are found in low numbers in bone marrow and are primarily quiescent. Retroviral integration and stable transduction of HSCs and their progeny require that the cells be in active cell cycle. A large fraction of the stem cells are in G0 phase of the cell cycle, that is, they are not actively cycling (Gordon et al. (1994) Leukemia 8:1068-1073). Efforts to optimize conditions for HSC transduction have focused on increasing the proportion of stem cells in cycle.
Cytokines have been used to induce cycling in cultured stem cells. For example, Reddy et al. (1995) Exp. Hematol. 23:813, reported that purified bone marrow stem/progenitor cells synchronously progressed from G0/G1 to S phase in vitro in response to a cytokine cocktail consisting of IL-3, IL-1.alpha., bFGF, GM-CSF, G-CSF CSF-1 and steel factor. Peters et al. (1995) Exp. Hematol. 23:461, described a cytokine cocktail of IL-3, IL-6, IL-11 and SCF used to expand murine hematopoietic progenitor cells in 48 hour in vitro culture of bone marrow. However, treatment with these and other cocktails of cytokines trigger differentiation of the cell; therefore, pluripotency is lost. IL-7 was found to inhibit proliferation in vitro of leukemic cells isolated from some acute lymphoblastic leukemia (ALL) by arresting the cells at late G1 but the growth inhibition was accompanied by maturation of the cells (Skonsberg et al. (1991) Blood 77:2445-2450). In another approach, mouse bone marrow cells were arrested in G1/G0 phase by culturing the cells in isoleucine-free medium (Reddy et al. (1995) Exp. Hematol. 23:813).
A preincubation of harvested bone marrow cells in cytokines is routinely used to enhance the retroviral transduction efficiency by stimulating the cells to enter active cell cycle. One such protocol for enhancing retroviral integration was described by Kittler et al. (1994) Blood 84:344A. The procedure involved prestimulating isolated murine bone marrow cells in medium containing a cocktail of cytokines for 48 hours, then co-culturing for an additional 48 hours in the same medium and cytokines with the retroviral producer cell line. The cells were then injected into host mice. This regimen however, produced low levels of engraftment in bone marrow and failed to achieve retroviral transduced cells in the bone marrow or in peripheral blood. Another procedure involved the pretreatment of mice with 5-fluorouracil (5-FU) and the addition of growth factors (IL-3, IL-6 or both) during or before the cocultivation of stem cells with virus producing cells prior to bone marrow transplantation (Bodine et al. (1991) Exp. Hematol. 19:206). However, 5-FU is a toxic drug.
The reported methods of triggering stem cells to enter the cell cycle either by the use of cytokines or other reagents have mostly been performed in vitro or ex vivo. Yet one study describes the use of a dekapeptide (pEEDCK).sub.2 injected into mice to trigger quiescent CFU-s (colony forming units) into the cell cycle, thus inducing a stem cell population expansion. (Paukovits et al. (1995) Cancer Research Therapy & Control, 4:203-209).
Thus, for long term success of hematopoietic stem cell gene therapy, there remains a need for efficient vector integration and maintenance of stem cell pluripotency. It is desirable to achieve a method of enriching or inducing for HSCs that are in active cycle without loss of pluripotency. In addition, the method should be applicable in vivo with minimal toxicity to the individual receiving treatment. The present invention satisfies these needs and provides related advantages as well.